This invention relates to vectors, in particular viral vectors such as retroviral vectors, which include heterologous, or foreign genes. More particularly, this invention relates to vectors including heterologous gene(s) and a negative selective marker.
Vectors are useful agents for introducing heterologous, or foreign, gene(s) or DNA into a cell, such as a eukaryotic cell. The heterologous, or foreign gene(s) is controlled by an appropriate promoter. In addition, the vector may further include a selectable marker, such as, for example, a neomycin resistance (neoR) gene, a hygromycin resistance (hygroR) gene, or a xcex2-galactosidase (xcex2-gal) gene, said marker also being under the control of an appropriate promoter. Examples of such vectors include prokaryotic vectors, such as bacterial vectors; eukaryotic vectors, including fungal vectors such as yeast vectors; and viral vectors such as DNA virus vectors, RNA virus vectors, and retroviral vectors. Retroviral vectors which have been employed for introducing heterologous, or foreign, genes or DNA into a cell include Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus and Harvey Sarcoma Virus. The term xe2x80x9cintroducingxe2x80x9d as used herein encompasses a variety of methods of introducing heterologous, or foreign, genes or DNA into a cell, such methods including transformation, transduction, transfection, and infection.
In accordance with an aspect of the present invention, there is provided a vector which includes a heterologous, or foreign gene, and a gene encoding a negative selective marker.
The vector which includes the heterologous, or foreign, gene, and the gene encoding a negative selective marker, may be a prokaryotic vector, such as a bacterial vector; a eukaryotic vector, such as a fungal vector, examples of which include yeast vectors; or a viral vector such as a DNA viral vector, an RNA viral vector, or a retroviral vector.
In a preferred embodiment, the vector is a viral vector, and in particular a retroviral vector. Examples of retroviral vectors which may be produced to include the heterologous gene and the gene encoding the negative selective marker include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, Avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumour virus.
Retroviral vectors are useful as agents to mediate retroviral-mediated gene transfer into eukaryotic cells. Retroviral vectors are generally constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the gene(s) of interest. Most often, the structural genes (i.e., gag, pol, and env), are removed from the retroviral backbone using genetic engineering techniques known in the art. This may include digestion with the appropriate restriction endonuclease or, in some instances, with Bal 31 exonuclease to generate fragments containing appropriate portions of the packaging signal.
These new genes have been incorporated into the proviral backbone in several general ways. The most straightforward constructions are ones in which the structural genes of the retrovirus are replaced by a single gene which then is transcribed under the control of the viral regulatory sequences within the long terminal repeat (LTR). Retroviral vectors have also been constructed which can introduce more than one gene into target cells. Usually, in such vectors one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter.
Efforts have been directed at minimizing the viral component of the viral backbone, largely in an effort to reduce the chance for recombination between the vector and the packaging-defective helper virus within packaging cells. A packaging-defective helper virus is necessary to provide the structural genes of a retrovirus, which have been deleted from the vector itself.
Bender et al., J. Virol. 61:1639-1649 (1987) have described a series of vectors, based on the N2 vector (Armentano, et al., J. Virol., 61:1647-1650) containing a series of deletions and substitutions to reduce to an absolute minimum the homology between the vector and packaging systems. These changes have also reduced the likelihood that viral proteins would be expressed. In the first of these vectors, LNL-XHC, there was altered, by site-directed mutagenesis, the natural ATG start codon of gag to TAG, thereby eliminating unintended protein synthesis from that point. In Moloney murine leukemia virus (MoMuLV), 5xe2x80x2 to the authentic gag start, an open reading frame exists which permits expression of another glycosylated protein (pPr80gag). Moloney murine sarcoma virus (MoMuSV) has alterations in this 5xe2x80x2 region, including a frameshift and loss of glycosylation sites, which obviate potential expression of the amino terminus of pPr80gag. Therefore, the vector LNL6 was made, which incorporated both the altered ATG of LNL-XHC and the 5xe2x80x2 portion of MoMuSV. The 5xe2x80x2 structure of the LN vector series thus eliminates the possibility of expression of retroviral reading frames, with the subsequent production of viral antigens in genetically transduced target cells. In a final alteration to reduce overlap with packaging-defective helper virus, Miller has eliminated extra env sequences immediately preceding the 3xe2x80x2 LTR in the LN vector (Miller et al., Biotechniques, 7:980-990, 1989).
The paramount need that must be satisfied by any gene transfer system for its application to gene therapy is safety. Safety is derived from the combination of vector genome structure together with the packaging system that is utilized for production of the infectious vector. Miller, et al. have developed the combination of the pPAM3 plasmid (the packaging-defective helper genome) for expression of retroviral structural proteins together with the LN vector series to make a vector packaging system where the generation of recombinant wild-type retrovirus is reduced to a minimum through the elimination of nearly all sites of recombination between the vector genome and the packaging-defective helper genome (i.e. LN with pPAM3).
In one embodiment, the retroviral vector may be a Moloney Murine Leukemia Virus of the LN series of vectors, such as those hereinabove mentioned, and described further in Bender, et al. (1987) and Miller, et al. (1989). Such vectors have a portion of the packaging signal derived from a mouse sarcoma virus, and a mutated gag initiation codon. The term xe2x80x9cmutatedxe2x80x9d as used herein means that the gag initiation codon has been deleted or altered such that the gag protein or fragments or truncations thereof, are not expressed.
In another embodiment, the retroviral vector may include at least four cloning, or restriction enzyme recognition sites, wherein at least two of the""sites have an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs; i.e., the restriction product has an average DNA size of at least 10,000 base pairs. Preferred cloning sites are selected from the group consisting of NotI, SnaBI, SalI, and XhoI. In a preferred embodiment, the retroviral vector includes each of these cloning sites.
When a retroviral vector including such cloning sites is employed, there may also be provided a shuttle cloning vector which includes at least two cloning sites which are compatible with at least two cloning sites selected from the group consisting of NotI, SnaBI, SalI, and XhoI located on the retroviral vector. The shuttle cloning vector also includes at least one desired gene which is capable of being transferred from the shuttle cloning vector to the retroviral vector.
The shuttle cloning vector may be constructed from a basic xe2x80x9cbackbonexe2x80x9d vector or fragment to which are ligated one or more linkers which include cloning or restriction enzyme recognition sites. Included in the cloning sites are the compatible, or complementary cloning sites hereinabove described. Genes and/or promoters having ends corresponding to the restriction sites of the shuttle vector may be ligated into the shuttle vector through techniques known in the art.
The shuttle cloning vector can be employed to amplify DNA sequences in prokaryotic systems. The shuttle cloning vector may be prepared from plasmids generally used in prokaryotic systems and in particular in bacteria. Thus, for example, the shuttle cloning vector may be derived from plasmids such as pBR322; pUC 18; etc.
The vectors of the present invention include one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and xcex2-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, TK promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
Heterologous or foreign genes which may be placed into the vectors of the present invention include, but are not limited to genes which encode cytokines or cellular growth factors, such as lymphokines, which are growth factors for lymphocytes. Other examples of foreign genes include, but are not limited to, genes encoding soluble CD4, Factor VIII, Factor IX, ADA, the LDL receptor, ApoE, and ApoC.
Suitable promoters which may control the foreign genes include those hereinabove described.
The vector also includes a gene encoding a suitable negative selective marker. An example of a negative selective marker is a gene which encodes thymidine kinase, or TK marker. It is to be understood, however, that the scope of the present invention is not to be limited to any specific negative selective marker or markers.
The vector of the present invention may be used to transduce packaging cells and to generate infectious viral particles. The infectious viral particles may be used to transduce cells (eg., eukaryotic cells such as mammalian cells). The vector containing the heterologous gene and the gene encoding the negative selective marker may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroportation, the use of liposomes, and CaPO4 precipitation. The eukaryotic cells may then be administered in vivo to a host as part of a gene therapy procedure. The eukaryotic cells which contain the infectious viral particles, as well as any other cells which are generated that contain the foreign gene and the gene encoding the negative selective marker, may be killed through the in vivo administration of an agent to a host. The selection of a suitable agent is dependent upon the heterologous gene and the gene encoding the negative selective marker contained in the vector.
For example, when the heterologous gene encodes a lymphokine, the negative selective marker may be a gene which encodes for thymidine kinase, or TK. When, for example, such a vector, which includes a heterologous gene encoding for a lymphokine, and a gene encoding for thymidine kinase as a negative selective marker, is employed to generate infectious viral particles, such infectious viral particles may be used to transfect tumor induced lymphocytes, or TIL cells. Such cells may then be administered to a host. The lymphokine may stimulate the production of tumor cells in the host. To prevent the further generation of cells containing the gene encoding the lymphokine and thus preventing the production of tumor cells, one may administer ganciclovir to the host. The ganciclovir may be administered in vivo and preferably by intravenous injection. In the presence of ganciclovir, such transfected tumor induced lymphocytes, and all other cells containing the gene encoding the lymphokine and the gene encoding thymidine kinase, are killed.
The vectors of the present invention may be produced from existing vectors through genetic engineering techniques known in the art such that the resulting vector includes a heterologous or foreign gene and a gene encoding a negative selective marker.
In accordance with an alternative aspect of the present invention, there is provided a vector system for the transduction of cells (e.g., eukaryotic cells and in particular mammalian cells) which comprises a first vector which includes a foreign gene, and a second vector which includes a gene encoding a negative selective marker. Each of the first and second vectors may be of the types hereinabove described, and may be constructed through genetic engineering techniques known to those skilled in the art. The foreign gene contained in the first vector may encode for those substances hereinabove described. The gene encoding the negative selective marker may be a gene encoding for thymidine kinase as hereinabove described, or any other negative selective marker. The first and second vectors may be used to tranduce packaging cells through techniques known in the art, and to generate infectious viral particles. The infectious viral particles generated from the first and second vectors may then be used to tranduce cells, such as eukaryotic, and in particular, mammalian cells, which then may be administered to a host as part of a gene therapy procedure.